Peterson Craig L, Hansen Jeffrey C
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
CSH Protoc. 2008 Dec 1;2008:pdb.prot5112. doi: 10.1101/pdb.prot5112.
INTRODUCTIONCore histones can be purified from a variety of cell sources, including Drosophila embryos, HeLa tissue culture cells, calf thymus, or chicken erythrocytes. Chick erythrocytes are an excellent source of cellular histones: Large quantities of source material are readily obtainable, the purified histones have low levels of post-translational modifications, and linker histones can also be purified from the same cell sample. Also, avian histones have an amino acid sequence identical to that of human histones. Histone stocks can be stored successfully for more than a year at 4°C and for several years at -20°C. With this protocol, 200 mL of blood usually yields in excess of 50 mg of purified histone octamers. Additional optional procedures are also presented for the purification of H1 and H5 linker histones, as well as for the preparation of H3/H4 tetramers and H2A/H2B dimers.
引言
核心组蛋白可从多种细胞来源中纯化得到,包括果蝇胚胎、HeLa组织培养细胞、小牛胸腺或鸡红细胞。鸡红细胞是细胞组蛋白的优质来源:易于获得大量的原材料,纯化后的组蛋白翻译后修饰水平较低,并且连接组蛋白也可从同一细胞样本中纯化得到。此外,鸟类组蛋白的氨基酸序列与人类组蛋白相同。组蛋白储备液可在4°C下成功储存一年以上,在-20°C下可储存数年。按照此方案,200 mL血液通常可产生超过50 mg的纯化组蛋白八聚体。还介绍了用于纯化H1和H5连接组蛋白以及制备H3/H4四聚体和H2A/H2B二聚体的其他可选步骤。